眼源性蜡样芽孢杆菌溶血素BL及其体外生物活性检测  

Identification of hemolysin BL from Bacillus cereus of patients with endophthalmitis and its biological activity detection in vitro

作　　者：郑美琴[1] 毛丽萍[1] 钟毓红[2] 潘娌妮[3] 李亚利[1] 楼永良[3] 

机构地区：[1]温州医学院附属眼视光医院检验科,325035 [2]浙江大学附属第二医院 [3]温州医学院检验医学院,325035

出　　处：《中华微生物学和免疫学杂志》2012年第12期1047-1052,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81171611）

摘　　要：目的对经API细菌鉴定条所证实的眼源性蜡样芽孢杆菌进行溶血素BL（HBL）基因鉴定并进行体外毒理性试验。方法根据溶血素结合亚基B、2个溶血亚基L1、L2三种组分的基因核苷酸序列，设计合成3对特异性引物，通过体系和条件优化，建立PCR检测方法，并对5株临床分离的眼源性蜡样芽孢杆菌进行检测分析；随机选取其中1株菌，利用盐析法提取溶血素，并以不同浓度剂量作用于绵羊红细胞、Vero细胞和Hela细胞，监测其对细胞的毒性；通过腹腔注射观察HBL对小鼠的致死作用，评估其毒力的强弱。结果5株临床分离菌中，共检出3个基因；蜡样芽孢杆菌溶血素BL蛋白具有较强溶血活性，其溶血活性呈明显的时间一剂量依赖性；BL溶血素对Vero细胞和Hela细胞的作用所致的形态变化虽有不同，但结果均导致细胞死亡，其内容物释出；对小鼠的致死率同样存在时间-剂量依赖性，且2．0HU／ml浓度以上的BL溶血素可使小鼠48h的致死率达100％。结论HBL具有较强的溶血活性，对临床分离的眼源性蜡样芽孢杆菌BL基因进行检测，3个基因均表达，为该菌感染后病情发展迅速、预后较差的临床表现提供了理论依据，同时为该病的快速诊断和分子流行病学调查提供了新的方法。Objective To identify the hemolysin BL（HBL） gene from Bacillus cereus of patients with endophthalmitis comfirmed by API bacterial identification test strip, and detect its biological activity in vitro. Methods Three pairs of specific primers were designed according to the gene sequence of HBL （ B, L1 and L2 components）, then the PCR assay were established through condition optimization, and to further detect five Bacillus cereus strains isolated from clinical patients with endophthalmitis. HBL with biological activity was extracted by salt fractionation from a randomly selected strain, and a series of different concentrations of HBL were prepared and acted on sheep red blood cells （SRBC）, Vero cells and Hela cells; virulence of HBL was also assessed through observating lethal effect of which on mice with intraperitoneal injection. Resuits Three genes of HBL were detected in all B. cereus strains from clinical patients; Strong hemolytic activity of HBL showed obvious timeand dose-dependent. In the study, we found the morphological changes of Vero and Hela cells caused by HBL were different, but cell death were the same result with contents released; Within 48 h after infection,lethality of HBL for mice showed 100% with the concentration of more than 2.0 HU/ml,and was also in a timeand dose-dependent manner. Conclusion HBL isolated from B. Cereus had high hemolysis activity and low concentration. The expression of all BL genes provided a strong basis for the clinical feature of B. Cereus infection, which was developed rapidly and with a poor prognosis. It also provided a new method for rapid diagnosis and molecular epidemiology investigation in clinical.

关 键 词：眼源性 蜡样芽孢杆菌 溶血素 

分 类 号：R440[医药卫生—诊断学]